Aims: Hypersensitivity pneumonitis of machinists associated with metalworking fluids (MWF) was recently linked to Mycobacterium immunogenum. In addition to Mycobacterium, impacts of continuous and massive contact to other micro-organisms, such as Pseudomonas, were little studied. This report intended to quantify and characterize the microbial load of 44 in-use MWF. Methods and Results: The main biodiversity of MWF was assessed using cultural methods, quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE). Total bacteria concentrations ranged from undetectable to 109 16S rRNA gene copies per millilitre. Concentrations obtained by qPCR were up to five orders of magnitude higher than by culture, suggesting that MWF contamination is generally underestimated. Two samples showed high concentrations of Myco. immunogenum (1 center dot 55 x 107 and 3 center dot 49 x 105 16S rRNA gene copies per millilitre). The overall biodiversity was low, as observed by culture and DGGE, and was comparable to data found in the literature. Pseudomonas pseudoalcaligenes was by far the main bacteria found in MWF samples (33 out of 44), followed by Ochrobactrum anthropi (32 out of 44). There was no significant relationship between the biodiversity profiles and the kind of MWF or equipment used, making it difficult to predict which micro-organisms will colonize each particular MWF. Conclusions: Very high concentrations of bacteria were found in most MWF studied and limited biodiversities were observed. Many species of micro-organisms were retrieved from MWF samples, but they were mostly colonized by Pseudomonas pseudoalcaligenes and Ochrobactrum anthropi. Significance and Impact of the Study: The major micro-organisms observed or recovered in this study from in-use MWF were present in very high concentrations, and thus further studies are needed to confirm their role in workers' respiratory disorders or health-related problems.